Cre-loxP recombination is an important tool in molecular genetics. Cre (“Causes recombination”) recombinase from bacteriophage P1 recognizes a specific 34 basepair (bp) target sequence, termed loxP, composed of an 8 bp spacer region flanked by two identical 13 bp inverted repeats (Table 1), e.g. Hoess et al Proc. Natl. Acad. Sci., 79: 3390-3402 (1982). Each base in the spacer region is conventionally named 1, 2, 3, 4, 5, 6, 7, or 8, according to its order (5′→3′) in the sequence. Cre-loxP sites mediate site specific intra- or inter-strand exchange of DNA molecules catalyzed by Cre recombinase. Depending on the location and the orientation of these sites, they can invert, insert, delete or exchange fragments of DNA in prokaryotic or eukaryotic systems, e.g. Sauer, Mol. Cell. Biol., 7: 2087-2096 (1987); Sauer et al, Proc. Natl. Acad. Sci., 85: 5166-5170 (1988); Sauer et al, Nucleic Acids Research, 17: 147-161 (1989). Orientation of insert DNA post-recombination is dependent on the orientation of the sites prior to the reaction, with sites in the same orientation on a given DNA strand mediating excision of intervening sequence and sites in opposite orientation mediating inversion of intervening sequence. Since the excision reaction is kinetically favored over the insertion reaction, gene deletion/inactivation experiments are straightforward to engineer by flanking the target sequence with loxP sites. The difficulty in implementing a stable DNA insertion is that the insertion reaction results in the presence of two loxP sites in cis configuration in the post-recombination product, which themselves become substrates for Cre and lead to rapid excision of the inserted component polynucleotide.
Two classes of variant loxP sites are available to engineer stable Cre-loxP integrative recombination. Both exploit sequence mutations in the Cre recognition sequence, either within the 8 bp spacer region or the 13-bp inverted repeats. Spacer mutants such as lox511, lox5171, lox2272, m2, m3, m7, and m11 recombine readily with themselves but have a markedly reduced rate of recombination with the wild-type site, e.g. Hoess et al, Nucleic Acids Research, 14: 2287-2300 (1986); Lee et al, Gene, 216: 55-65 (1998); Langer et al, Nucleic Acids Research, 30: 3067-3077 (2002). This class of mutants has been exploited for DNA insertion by Recombinase Mediated Cassette Exchange (RMCE), e.g. Seibler et al, Biochemistry, 36: 1740-1747 (1997); Schlake et al, Biochemistry, 33: 12746-12751 (1994); Baer et al, Curr. Opin. Biotech., 12: 473-480 (2001). Inverted repeat mutants represent the second class available and contain altered bases in the left inverted repeat (LE mutant) or the right inverted repeat (RE mutant). The LE mutant, lox71, has 5 bp on the 5′ end of the left inverted repeat that are changed from the wild type sequence to TACCG, e.g. Albert et al, Plant J., 7: 649-659 (1995); Araki et al, Nucleic Acids Research, 25: 868-872 (1997). Similarly, the RE mutant, lox66, has the five 3′-most bases changed to CGGTA. Inverted repeat mutants are used for integrating plasmid inserts into chromosomal DNA with the LE mutant designated as the “target” chromosomal loxP site into which the “donor” RE mutant recombines. Post-recombination, loxP sites are located in cis, flanking the inserted component polynucleotide. The mechanism of recombination is such that post-recombination one loxP site is a double mutant (containing both the LE and RE inverted repeat mutations) and the other is wild type, e.g. Van Duyne et al, Annu. Rev. Biophys. Biomol. Struct., 30: 87-104 (2001); Lee et al, Prog. Nucleic Acid Res. Mol. Biol., 80: 1-42 (2005); Lee et al, J. Mol. Biol., 326: 397-412 (2003). The double mutant is sufficiently different from the wild-type site that it is unrecognized by Cre recombinase and the inserted component polynucleotide is not excised. Recently, spacer and inverted repeat mutants have been combined to increase the specificity and stability of integrative recombination, e.g. Langer et al (cited above); Araki et al, Nucleic Acids Research, 30: e103 (2002).
Previously, novel spacer mutants have been discovered by mutating bases or by generating a set of potential spacer mutants and testing recombination between these spacers with the wild-type loxP site, e.g. Langer et al (cited above); Lee et al (Gene, cited above). In particular, Lee et al used an in vitro assay that evaluated the recombination efficiency of 24 spacers with 1 bp substitutions and 30 spacers with 2 bp substitutions from the sequence of the wild-type loxP. Their data suggested that homology was required at positions 2-5 and positions 6-7 for efficient strand exchange and resolution of a Holiday junction, whereas positions 1 and 8 had relaxed homology requirements. They concluded that homology was essential to achieve recombination rates between mutant loxP spacers comparable to that of the wild-type sequence. Their success with the lox2272 mutant suggested that positions 2 and 7 were particularly important in blocking promiscuous recombination.
The above work is important because recombination systems, such Cre-loxP and others, provide a means for making and/or manipulating large polynucleotide constructs that are useful in fields, such as synthetic biology, metabolic engineering, and the like, where practitioners seek to improve cellular activities by large-scale manipulation of enzymatic, transport, and regulatory functions of cells, e.g. Bailey, Science, 252: 1668-1674 (1991). It would be highly useful to such fields if there were available additional recombination elements that could be used together without cross-reactivity for the purpose of constructing large polynucleotide constructs, particularly through successive cycles of site-specific recombination.